Biomolecular condensates have emerged as major drivers of cellular organization. It remains largely unexplored, however, whether these condensates can impart mechanical function(s) to the cell. The heterochromatin protein HP1α (Swi6 in Schizosaccharomyces pombe) crosslinks histone H3K9 methylated nucleosomes and has been proposed to undergo condensation to drive the liquid-like clustering of heterochromatin domains. Here, we leverage the genetically tractable S. pombe model and a separation-of-function allele to elucidate a mechanical function imparted by Swi6 condensation. Using single-molecule imaging, force spectroscopy, and high-resolution live-cell imaging, we show that Swi6 is critical for nuclear resistance to external force. Strikingly, it is the condensed yet dynamic pool of Swi6, rather than the chromatin-bound molecules, that is essential to imparting mechanical stiffness. Our findings suggest that Swi6 condensates embedded in the chromatin meshwork establish the emergent mechanical behavior of the nucleus as a whole, revealing that biomolecular condensation can influence organelle and cell mechanics.

Cell wall integrity (CWI) is crucial for the growth, development, and host invasion of pathogenic fungi. The APSES transcription factor Swi6 in fungi plays a role in mediating cell wall integrity through the mitogen-activated protein kinase (MAPK) signaling pathway. Ceratocystis fimbriata is a notorious pathogenic fungus responsible for causing black rot in sweet potatoes. In this study, an orthologous APSES transcription factor Swi6 (CfSwi6) downstream of the CWI regulatory pathway in C. fimbriata was characterized. Deletion of CfSWI6 leads to impaired hyphal development, conidiation, and compromised cell wall integrity, resulting in a significant reduction in virulence. Transcriptome analysis revealed the involvement of CfSWI6 in various pathways, including the MAPK pathway, DNA synthesis and stress response. ChIP-seq data provided predictions of potential target genes regulated by CfSwi6. Through yeast one-hybrid, we confirmed the direct binding of CfSwi6 to the promoter of the chitin synthetase gene. In summary, these findings indicated that CfSwi6 plays an important role in the growth, development, and pathogenicity of C. fimbriata. This study provides new insights into the pathogenic mechanism of C. fimbriata in sweet potato and inspires potential strategies to control sweet potato black rot.

Ceratocystis fimbriata (C. fimbriata) is a notorious pathogenic fungus that causes sweet potato black rot disease. The APSES transcription factor Swi6 in fungi is located downstream of the cell wall integrity (CWI)-mitogen-activated protein kinase (MAPK) signaling pathway and has been identified to be involved in cell wall integrity and virulence in several filamentous pathogenic fungi. However, the specific mechanisms by which Swi6 regulates the growth and pathogenicity of plant pathogenic fungi remain elusive. In this study, the SWI6 deletion mutants and complemented strains of C. fimbriata were generated. Deletion of Swi6 in C. fimbriata resulted in aberrant growth patterns. Pathogenicity assays on sweet potato storage roots revealed a significant decrease in virulence in the mutant. Non-targeted metabolomic analysis using LC-MS identified a total of 692 potential differentially accumulated metabolites (PDAMs) in the ∆Cfswi6 mutant compared to the wild type, and the results of KEGG enrichment analysis demonstrated significant enrichment of PDAMs within various metabolic pathways, including amino acid metabolism, lipid metabolism, nucleotide metabolism, GPI-anchored protein synthesis, and ABC transporter metabolism. These metabolic pathways were believed to play a crucial role in mediating the growth and pathogenicity of C. fimbriata through the regulation of CWI. Firstly, the deletion of the SWI6 gene led to abnormal amino acid and lipid metabolism, potentially exacerbating energy storage imbalance. Secondly, significant enrichment of metabolites related to GPI-anchored protein biosynthesis implied compromised cell wall integrity. Lastly, disruption of ABC transport protein metabolism may hinder intracellular transmembrane transport. Importantly, this study represents the first investigation into the potential regulatory mechanisms of SWI6 in plant filamentous pathogenic fungi from a metabolic perspective. The findings provide novel insights into the role of SWI6 in the growth and virulence of C. fimbriata, highlighting its potential as a target for controlling this pathogen.

DNA is stored in the nucleus of a cell in a folded state; however, only the necessary genetic information is extracted from the required group of genes. The key to extracting genetic information is chromatin ambivalence. Depending on the chromosomal region, chromatin is characterized into low-density \"euchromatin\" and high-density \"heterochromatin\", with various factors being involved in its regulation. Here, we focus on chromatin regulation and gene expression by the yeast FACT complex, which functions in both euchromatin and heterochromatin. FACT is known as a histone H2A/H2B chaperone and was initially reported as an elongation factor associated with RNA polymerase II. In budding yeast, FACT activates promoter chromatin by interacting with the transcriptional activators SBF/MBF via the regulation of G1/S cell cycle genes. In fission yeast, FACT plays an important role in the formation of higher-order chromatin structures and transcriptional repression by binding to Swi6, an HP1 family protein, at heterochromatin. This FACT property, which refers to the alternate chromatin-regulation depending on the binding partner, is an interesting phenomenon. Further analysis of nucleosome regulation within heterochromatin is expected in future studies.

BACKGROUND: Swi6 acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. This study investigates how nucleocytoplasmic transport by multiple transport factors may influence specific Swi6 activities.
RESULTS: Here we show that the exportin Crm1 is important for Swi6 nuclear export and activity. Loss of a putative Crm1 NES or inhibition of Crm1 activity results in changes in nucleocytoplasmic Swi6 localization. Alteration of the Crm1 NES in Swi6 results in decreased MBF-mediated gene expression, but does not affect SBF reporter expression, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the putative Crm1 NES in Swi6 results in cells that are larger than wild type, and this increase in cell size is exacerbated by deletion of Msn5.
CONCLUSIONS: These data provide evidence that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal. We identify a putative nuclear export signal for Crm1 within Swi6, and observe that export by Crm1 or Msn5 independently influences Swi6-regulated expression of a different subset of Swi6-controlled genes. These findings provide new insights into the complex regulation of Swi6 transcription activation activity and the role of nucleocytoplasmic shuttling in regulated gene expression.

Budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe are good models for heterochromatin study. In S. pombe, H3K9 methylation and Swi6, an ortholog of mammalian HP1, lead to heterochromatin formation. However, S. cerevisiae does not have known epigenetic silencing markers and instead has Sir proteins to regulate silent chromatin formation. Although S. cerevisiae and S. pombe form and maintain heterochromatin via mechanisms that appear to be fundamentally different, they share important common features in the heterochromatin structural proteins. Heterochromatin loci are localized at the nuclear periphery by binding to perinuclear membrane proteins, thereby producing distinct heterochromatin foci, which sequester heterochromatin structural proteins. In this review, we discuss the nuclear peripheral anchoring of heterochromatin foci and its functional relevance to heterochromatin formation and maintenance.

Molecular signal transduction networks, which conduct transcription at the G1 to S phase transition of the eukaryotic cell division cycle have been identified in diverse taxa from mammals to baker\'s yeast with analogous functional organization. However, regarding some network components, such as the transcriptional regulators STB1 and WHI5, only few orthologs exist, which are confined to individual Saccharomycotina species. While Whi5 has been characterized as yeast analog of human Rb protein, in the particular case of Stb1 (Sin three binding protein 1) identification of functional analogs emerges as difficult because to date its exact functionality still remains obscured. By aiming to resolve Stb1\'s enigmatic role this Perspective article especially surveys works covering relations between Cyclin/CDKs, the heteromeric transcription factor complexes SBF (Swi4/Swi6) and MBF (Mbp1/Swi6), as well as additional coregulators (Whi5, Sin3, Rpd3, Nrm1) which are collectively associated with the orderly transcription at \'Start\' of the Saccharomyces cerevisiae cell cycle. In this context, interaction capacities of the Sin3-scaffold protein are widely surveyed because its four PAH domains (Paired Amphiphatic Helix) represent a \'recruitment-code\' for gene-specific targeting of repressive histone deacetylase activity (Rpd3) via different transcription factors. Here, Stb1 plays a role in Sin3\'s action on transcription at the G1/S-boundary. Through bioinformatic analyses a potential Sin3-interaction domain (SID) was detected in Stb1, and beyond that, connections within the G1/S-regulatory network are discussed in structural and evolutionary context thereby providing conceptual perspectives.

FACT (facilitate chromatin transcription) is involved in heterochromatic silencing, but its mechanisms and function remain unclear. We reveal that the Spt16 recruitment mechanism operates in two distinct ways in heterochromatin. First, Pob3 mediates Spt16 recruitment onto the heterochromatin through its Spt16 dimerization and tandem PH domains. Without Pob3, Spt16 recruitment is partially reduced, exhibiting a silencing defect and impaired H2A/H2B organization. Second, heterochromatin protein 1 (HP1)/Swi6 mediates Spt16 recruitment onto the heterochromatin by physical interaction of the Swi6 chromo-shadow domain (CSD) and Spt16 peptidase-like domains. Several CSD mutants are tested for Spt16 binding activity, and the charged loop connecting β1 and β2 is critical for Spt16 binding and heterochromatic silencing. Loss of these pathways causes a severe defect in H3K9 methylation and HP1/Swi6 localization in the pericentromeric region, exhibiting transcriptional silencing defects and disordered heterochromatin. Our findings suggest that FACT and HP1/Swi6 work intimately to regulate heterochromatin organization.

Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.

The fission yeast centromere, which is similar to metazoan centromeres, contains highly repetitive pericentromere sequences that are assembled into heterochromatin. This is required for the recruitment of cohesin and proper chromosome segregation. Surprisingly, the pericentromere replicates early in the S phase. Loss of heterochromatin causes this domain to become very sensitive to replication fork defects, leading to gross chromosome rearrangements. This review examines the interplay between components of DNA replication, heterochromatin assembly, and cohesin dynamics that ensures maintenance of genome stability and proper chromosome segregation.